CN110301455B - 阿孙链霉菌产生的挥发性物质在植物病害防治中的应用 - Google Patents
阿孙链霉菌产生的挥发性物质在植物病害防治中的应用 Download PDFInfo
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Abstract
本发明公开了阿孙链霉菌产生的挥发性物质在植物病害防治中的应用,具体公开阿孙链霉菌产生的挥发性物质能够抑制荔枝霜疫霉等多种病原菌物的生长,抑制率达63~100%。本发明还进一步明确了阿孙链霉菌产生的挥发性物质中14种具有明显抑菌作用的化合物,为今后研究荔枝霜疫霉等植物病原菌的防治药物提供思路。
Description
技术领域
本发明涉及植物病害生物防治领域,尤其涉及阿孙链霉菌产生的挥发性物质在植物病害防治中的应用。
背景技术
挥发性物质是一类低分子量(300Da)的化合物,非极性,易挥发,蒸汽压较高,室温下可以达到0.01kPa(Morath et al.,2012)。早在1973年Moore-Landecker&Stotzky就报道了放线菌产生的挥发性物质导致曲霉分生孢子萎缩和青霉菌、镰刀菌、绿色木霉等菌液泡增大。Strobel等(2001)人报道内生真菌Muscodor albus产生的挥发性物质通过熏蒸法可防治多种采后病害(Mercier,2004;Mercier et al.,2005)和土传病害。近年来,越来越多的科研工作者从事微生物挥发性物质的研究。万明国(2008)报道了在密闭容器中普特拉链霉菌能有效的抑制草莓灰霉病、水稻纹枯病、油菜菌核病的发生。球孢链霉菌JK-1产生的挥发性物质导致灰霉病菌、柑橘青霉菌菌丝畸形、细胞膜受到破坏,抑制菌丝生长、孢子萌发以及分生孢子形成,并且可有效控制柑橘青霉病的发生(李其利,2011),张清华(2014)报道了CanR-46是油菜的内生真菌,其产生挥发性的物质可以抑制核盘菌的生长。
可见,微生物产生的挥发性物质在病原菌防治方面的研究已经取得一定的成果。但目前对于阿孙链霉菌挥发性物质的研究相对较少,研究人员尚不明确阿孙链霉菌在植物病害防治方面的重要作用,亟需深入研究。
发明内容
本发明提供一种阿孙链霉菌产生的挥发性物质在植物病害防治中的应用,有效填补阿孙链霉菌在植物病害防治方面的技术空白。
本发明采取的技术方案如下:
本发明提供了一种阿孙链霉菌产生的挥发性物质在植物病害防治中的应用,所述植物病害为由荔枝霜疫霉、辣椒疫霉、芋疫霉、群结腐霉、链格孢属、小麦赤霉病菌、香蕉炭疽病菌、水稻稻瘟病菌、葡萄溃疡病菌或番木瓜疮痂病菌引起的植物病害。所述挥发性物质对于荔枝霜疫霉菌丝生长、孢子囊产量和卵孢子产量有明显的抑制作用。
本发明还进一步通过GC-MS分析,明确了挥发性物质的成分包括:亚砷酸三(三甲基硅基)酯、2-甲基异茨醇、八甲基环四硅氧烷、十三烷、二甲基二十四烷、苯甲酸己酯、2,6,10-甲基十二烷、十四烷、顺式-7,8-环氧-2-甲基十八烷、2,6,10-甲基十三烷、正二十八烷、正十五烷、正十六烷和长叶烯。
所述的挥发性物质的制备方法,包括以下步骤:
S1:将阿孙链霉菌菌株在PDA平板上划线培养7d,刮取新鲜孢子接入ISP2培养基后,置于摇床上振荡培养,然后制备孢子悬浮液,接种到小麦粒培养基上培养,获得阿孙链霉菌麦粒培养物;
S2:将麦粒培养物放入小瓶中,密封放置后,萃取。
更进一步的,步骤S1为:将阿孙链霉菌菌株在PDA平板上划线培养7d,刮取新鲜孢子接入含有100mL ISP2培养基的三角瓶内,然后置于摇床上,以200rpm、28℃振荡培养3d,制备浓度1×107个孢子/mL的孢子悬浮液,按1mL/100g的比例接种到小麦粒培养基上,摇匀后,置于25℃条件下培养20d,其间每3d将麦粒培养基摇匀一次。
步骤S2为:称取培养20d的阿孙链霉菌麦粒培养物放入瓶中,密封后25℃下放置12h,然后用SPME萃取头萃取。
与现有技术相比,本发明的有益效果是:
(1)阿孙链霉菌产生的挥发性物质能够抑制荔枝霜疫霉等多种病原菌物的生长,抑制率达63~100%。
(2)明确了阿孙链霉菌产生的挥发性物质中14种具有明显抑菌作用的化合物,为今后研究荔枝霜疫霉的防治药物提供方向。
(3)明确了阿孙链霉菌产生的挥发性物质对于荔枝霜疫霉菌丝生长、孢子囊产量和卵孢子产量均有明显的抑制作用。
附图说明
图1为不同培养时间对阿孙链霉菌TJGA-19产生挥发性物质抑菌活性影响效果图;图中a,b,c,d分别为对照和阿孙链霉菌TJGA-19在小麦培养基上培养10d、20d、30d的培养物的处理;
图2为不同培养时间对阿孙链霉菌TJGA-19产生挥发性物质抑菌活性影响结果统计图;
图3为不同量的阿孙链霉菌TJGA-19小麦粒培养物产生的挥发性物质对荔枝霜疫霉菌丝生长的影响效果图;图中,a,b,c,d,e,f,g,h,i分别为对照和2g/L、4g/L、8g/L、12g/L、16g/L、24g/L、32g/L、40g/L的阿孙链霉菌TJGA-19小麦粒培养物处理;
图4为不同量的阿孙链霉菌TJGA-19小麦粒培养物产生的挥发性物质对荔枝霜疫霉菌丝生长的影响结果统计图;
图5为不同量的阿孙链霉菌TJGA-19小麦粒培养物产生的挥发性物质对荔枝霜疫霉孢子囊产量的影响结果统计图;
图6为不同量的阿孙链霉菌TJGA-19小麦粒培养物产生的挥发性物质对卵孢子产量的影响效果图;a,b,c,d,e,f,g,h,i,j,k分别为对照和3g/L、4g/L、6g/L、8g/L、12g/L、16g/L、24g/L、32g/L、40g/L的阿孙链霉菌TJGA-19小麦粒培养物处理;
图7为不同量的阿孙链霉菌TJGA-19小麦粒培养物产生的挥发性物质对卵孢子产量的影响结果统计图;
图8为扫描电镜观察挥发性物质对荔枝霜疫霉形态的影响效果图;a,b,c为对照;d,e,f为100g/L的阿孙链霉菌TJGA-19小麦粒培养物处理。
图9为透射电镜观察挥发性物质对荔枝霜疫霉菌丝亚细胞结构的影响效果图;a,b,c为对照;d,e,f为100g/L的阿孙链霉菌TJGA-19小麦粒培养物处理。a,d为孢子囊纵向切面;b,e为菌丝横向切面;c,f为菌丝纵向切面;N:细胞核;W:细胞壁;
图10为挥发性物质对离体叶片荔枝霜疫病的防效效果图;a,b,c,d,e分别为对照和8g/L、16g/L、24g/L、32g/L的阿孙链霉菌TJGA-19小麦粒培养物处理;
图11为挥发性物质对离体叶片荔枝霜疫病的防效结果统计图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
本发明实施例所用的实验方法如无特殊说明,均为常规方法。
本发明实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明实施例实验接种所用的荔枝叶片选取的荔枝品种为怀枝。
本发明实施例所用的荔枝叶片采自华南农业大学园艺学院实验基地荔枝果园,选取幼嫩、形状、大小、叶龄一致,无病虫害、无机械损伤的叶片,接种前用流水冲洗干净,室温风干。
本发明所述阿孙链霉菌TJGA-19(Streptomyces abikoensis TJGA-19),由海南大学植物保护学院植物病害生物防控实验室分离保存,可由现有技术中的常规方法获得。
本发明实施例8所述荔枝霜疫霉、辣椒疫霉、芋疫霉等病原菌为海南大学植物保护学院植物病害生物防控实验室分离保存,可由现有技术中的常规方法获得。
本发明实施例所述小麦粒培养基:将小麦加足水煮沸至表皮开裂后过滤,晾干,分装灭菌。
实施例1、阿孙链霉菌TJGA-19产生挥发性物质的制备
将菌株TJGA-19在PDA平板上划线培养7天,刮取新鲜孢子接入含有100mL ISP2培养基的250mL三角瓶内,然后置于200rpm的摇床上28℃振荡培养3d,制备孢子悬浮液(约1×107个孢子/mL),按1mL/100g(V/W)的比例接种量接种到无菌的小麦粒培养基上。摇匀后,置于25℃条件下培养20d,其间每3d将麦粒培养基摇匀一次,以便菌株TJGA-19均匀生长。
实施例2、培养时间对阿孙链霉菌TJGA-19产生挥发性物质抑菌活性的影响
在大培养皿中(h=30mm,内部总体积500mL)放置四个小培养皿底 其中3个小培养皿含约5mL的PDA培养基,并在培养基中央接种一块荔枝霜疫霉菌饼另外一个小培养皿放置40g/L阿孙链霉菌TJGA-19小麦粒培养物(25℃条件下分别培养了10d、20d、30d),以40g/L的无菌小麦粒为对照,然后将大培养皿密封。在25℃恒温培养6d,测量菌落直径并计算抑菌率。每个处理3个重复,实验重复3次。结果见图1和图2。
菌丝生长抑制率(%)=(对照菌落直径-处理菌落直径)/对照菌落直径×100
结果表明:
培养时间不同,阿孙链霉菌TJGA-19产生的挥发性物质抑菌活性存在较大的差异。阿孙链霉菌TJGA-19在小麦粒培养基上培养20d产生的挥发性物质抑菌活性最强,抑菌率达74.1%,而培养10d和30d的处理的抑菌率分别为54.9%和63.7%,显著低于培养20d处理的抑菌率。
实施例3、挥发性物质的收集及鉴定
称取培养20d的阿孙链霉菌TJGA-19小麦粒培养物4g放入20mL小瓶中,用锡铂纸密封后25℃下放置12h,然后用SPME萃取头扦入瓶中,推出纤维头[metal alloy(PDMS 100μm)]吸附30min后收回纤维头,将萃取头手动进样到GC-MS(Agilent 7890B American),250℃下解吸附3min,进行GC-MS分析。GC-MS操作条件参照(Strobel,et al.2001)。计算机自动将所得到的气体成分质谱与国际标准数据库(NIST 14Mass Spectrometry Librarydatabases)数据进行比对,鉴定出挥性物质的成分。以无菌空白等量小麦为对照,将对照与阿孙链霉菌TJGA-19小麦粒培养物中同时存在的物质去掉。每个处理3个重复。
经GC-MS检测培养20d的阿孙链霉菌TJGA-19产生的挥发性物质,获得14种化合物(表1)。这些化合物多数分属于烯类、酯类、有机酸、烷烃等。
表1
实施例4、挥发性物质对荔枝霜疫霉菌丝生长及孢子囊产量的影响
将4个小培养皿底放置在一个大培养皿中,其中3个小培养皿含约5mL的PDA培养基,在培养基中央接种一块荔枝霜疫霉菌饼另外一个小培养皿放置实施例1的阿孙链霉菌TJGA-19小麦粒培养物,用封口膜密封大培养皿,25℃恒温培养6d,测量菌落直径,然后无菌水冲洗菌落表面,滤去菌丝,统计孢子囊产量。实验设置8个处理,阿孙链霉菌TJGA-19小麦粒培养物的量分别为2g/L、4g/L、8g/L、12g/L、16g/L、24g/L、32g/L、40g/L,以等量无菌空白小麦粒为对照。每个处理3个重复,实验重复3次。结果见图3-图6。
实验结果表明:小麦粒培养物的量越大,抑菌活性越强。在25℃下培养6d,对照的菌落直径47.3mm,培养物的量为2g/L、4g/L、8g/L时,菌落直径分别为46.8mm和46.2mm和45.4mm,它们与对照相比无显著性差异,当培养物的量为12g/L、16g/L、24g/L、32g/L、40g/L时,菌落直径分别为39.6mm、33.2mm、26.3mm、20.6mm、1.22mm,显著低于对照的菌落直径。
在25℃下培养6d,对照的孢子囊产量为129.2×104个孢子囊/皿,培养物的量为2g/L、4g/L时,孢子囊产量为124.7×104、123.5×104个孢子囊/皿,与对照相比无显著性差异,在培养物的量为8g/L、12g/L、16g/L、24g/L的处理中,孢子囊产量分别为78×104、51.3×104、5.3×104和2.6×104个孢子囊/皿,在培养物的量为32g/L、40g/L的处理中,没有孢子囊形成。
实施例5挥发性物质对荔枝霜疫霉卵孢子产量的影响
将4个小培养皿底放置在一个大培养皿中,其中3个小培养皿含约3mL的CA培养基,在平板中央放置一块荔枝霜疫霉菌饼另外一个小培养皿放置实施例1的阿孙链霉菌TJGA-19小麦粒培养物,用封口膜密封大培养皿,25℃恒温黑暗培养14d,在离接种点10mm的区域内随机切取三块菌丝块置于10mL离心管中,加入3mL超纯水,用高速匀浆机(6000rpm)匀浆2min,然后在显微镜下统计50μL卵孢子数量,从而求得单位面积的卵孢子数量(Flier et al.,2001)。实验设置10个处理,阿孙链霉菌TJGA-19小麦粒培养物的量分别为3g/L、4g/L、6g/L、8g/L、12g/L、16g/L、24g/L、32g/L和40g/L,以等量无菌空白小麦粒为对照。每个处理3个重复,实验重复3次。
单位面积的卵孢子产量(个/cm2)=50μL悬浮液中的卵孢子数量×60/2.36
实验结果表明:在25℃黑暗条件下培养14d,在对照组及3g/L的处理组均产生大量的卵孢子,分别为2686.4个/cm2、2669.5个/cm2;当培养物的量为4g/L时,卵孢子产量为2347.4个/cm2,与对照相比显著下降;当培养物的量为6g/L、8g/L时,卵孢子产量急剧下降到1466.1个/cm2、57.3个/cm2;当培养物的量增加至12g/L、16g/L、24g/L、32g/L、40g/L时,没有卵孢子形成(图7)。
实施例6挥发性物质对荔枝霜疫霉超微结构的影响
先将荔枝霜疫霉接种在CA培养基上,置于25℃培养3天,再与装有100g/L阿孙链霉菌TJGA-19小麦粒培养物(实施例1方法制得)的培养皿对扣,密封,继续培养3天,然后从菌落边缘切取约8mm×5mm×3mm菌块,每个处理切取3块菌块,立即投入预冷的4%的戊二醛固定液,4℃低温过夜,用于扫描电镜制样;用无菌牙签轻轻刮取菌丝体放入预冷的2.5%戊二醛中预固定,用于透射电镜制样。以等量无菌空白小麦粒为对照。
扫描电镜制样:样品在4%的戊二醛固定液(用0.1mol·L-1pH为7.2的磷酸缓冲液配制)中4℃低温过夜,用0.1mol·L-1pH为7.2的磷酸缓冲液漂洗3次,每次15min,1%锇酸后固1h,依次用浓度为30%、50%、70%、80%和90%的酒精脱水,每次10min,接着用100%酒精脱水两次,每次10min,最后用醋酸异戊酯过渡两次,每次15min,临界点干燥,粘样,渡膜后用LEO-1530VP扫描电镜,在5KV下观察并拍照。
透射电镜制样:样品在2.5%的戊二醛固定液(用0.1mol·L-1p H为7.2的磷酸缓冲液配制)中4℃低温过夜,用0.1mol·L-1pH为7.2的磷酸缓冲液漂洗3次,每次15min,1%锇酸后固,4℃低温过夜,酒精脱水,最后用环氧树脂渗透,包埋,超薄切片机(EM UC7,Leica)切成70nm超薄切片,醋酸铀柠檬酸铅双染色,透射电镜(Tecnai,FEI)在100kV进行菌丝细胞内部结构的观察并拍照。
扫描电镜结果显示,在对照处理中荔枝霜疫霉菌丝体均匀、细长、表面光滑、饱满,孢子囊球形、饱满、胞壁光滑(图8a,b,c);当荔枝霜疫霉菌丝体暴露在100g/L阿孙链霉菌TJGA-19小麦粒培养物产生的挥发性气体中,菌丝体塌陷、干瘪,孢子囊细胞壁粗糙、下陷(图8d,e,f)。透射电镜结果显示,对照孢子囊细胞质均匀,细胞器排列整,细胞核形态正常(图9a,b,c);在100g/L的麦粒培养物处理中线粒体、细胞核消失,液泡增多、增大(图9d,e,f)。
实施例7挥发性物质对离体叶片荔枝霜疫病的防治效果
在无菌大培养皿(h=30mm)的底部铺上一层滤纸在滤纸上喷8mL无菌水保湿,在滤纸中央放置一个直径90mm的培养皿底,培养皿中装有不同数量的阿孙链霉菌TJGA-19小麦粒培养物(实施例1方法制得),然后将10片叶片铺在滤纸外缘,叶背朝上,取2μL孢子囊悬浮液(2×104个孢子囊/mL)滴在片脉上,立即密封大培养皿。置于25℃条件下培养48h后统计病斑长度。实验设置4个处理,阿孙链霉菌TJGA-19小麦粒培养物的量分别为8g/L、16g/L、24g/L、32g/L,以等量无菌空白小麦粒为对照。每个处理30片叶片,每个处理3个重复,共90片叶片,实验重复3次。结果见图10-图11。
结果表明:阿孙链霉菌TJGA-19产生的挥发性物质对离体叶片荔枝霜疫病有很好的防效。接种后48h,对照的病斑直径为26.7mm,当阿孙链霉菌TJGA-19小麦粒培养物的量从8g/L提高到32g/L,病斑直径从21.9mm下降到3mm。
实施例8阿孙链霉菌TJGA-19产生的挥发性物质的抑菌谱测定
将供试病原菌接种到PDA培养基上,25℃恒温培养箱中培养6d然后用打孔器在菌落外缘制取菌饼,置于新鲜PDA平板中央,称取32g/L阿孙链霉菌TJGA-19小麦粒培养物(制备方法参见实施例1)移入90mm培养皿中,然后将两皿底相对扣在一起,密封,做成双皿对扣的装置,病原菌在上,小麦粒培养物在下,以32g/L的无菌小麦粒为对照。将双皿对扣装置放置在25℃培养箱中培养,每天观察,待各病原菌对照菌落长满培养皿时测量抑菌圈的直径(十字交叉法),计算抑制率,每个处理3个重复,实验重复3次。结果见表2。
菌丝生长抑制率(%)=(对照菌落直径-处理菌落直径)/对照菌落直径×100%
表2:阿孙链霉菌TJGA-19产生的挥发性物质的抑菌谱
实验结果表明阿孙链霉菌TJGA-19产生的挥发性物质能够抑制多种植物病原菌的生长,其中对荔枝霜疫霉、辣椒疫霉、芋疫霉、群结腐霉、链格孢属、小麦赤霉病菌、香蕉炭疽病菌、水稻稻瘟病菌、葡萄溃疡病菌和番木瓜疮痂病菌的抑制作用最强,抑制率达63~100%。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换。
Claims (3)
1.阿孙链霉菌产生的挥发性物质在植物病害防治中的应用,其特征在于,所述植物病害为由辣椒疫霉或芋疫霉引起的植物病害;所述挥发性物质由以下成分组成:亚砷酸三(三甲基硅基)酯、2-甲基异茨醇、八甲基环四硅氧烷、十三烷、二甲基二十四烷、苯甲酸己酯、2,6,10-甲基十二烷、十四烷、顺式-7,8-环氧-2-甲基十八烷、2,6,10-甲基十三烷、正二十八烷、正十五烷、正十六烷和长叶烯;
所述挥发性物质的制备方法包括以下步骤:
S1:将阿孙链霉菌菌株在PDA平板上划线培养7d,刮取新鲜孢子接入ISP2培养基后,置于摇床上振荡培养,然后制备孢子悬浮液,接种到小麦粒培养基上培养,获得阿孙链霉菌麦粒培养物;
S2:将麦粒培养物放入小瓶中,密封放置后,用SPME萃取头萃取。
2.根据权利要求1所述的阿孙链霉菌产生的挥发性物质在植物病害防治中的应用,其特征在于,所述步骤S1中:将阿孙链霉菌菌株在PDA平板上划线培养7d,刮取新鲜孢子接入含有100mL ISP2培养基的三角瓶内,然后置于摇床上,以200rpm、28℃振荡培养3d,制备浓度1×107个孢子/mL的孢子悬浮液,按1mL/100g的比例接种到小麦粒培养基上,摇匀后,置于25℃条件下培养20d,其间每3d将麦粒培养基摇匀一次。
3.根据权利要求1所述的阿孙链霉菌产生的挥发性物质在植物病害防治中的应用,其特征在于,步骤S2:称取培养20d的阿孙链霉菌麦粒培养物放入瓶中,密封后25℃下放置12h,然后用SPME萃取头萃取。
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